Aquatic Birnaviruses such as infectious pancreatic necrosis virus (IPNV) are the causal agent of a highly contagious and destructive disease of juvenile Rainbow and Brook trout, and Atlantic salmon (Wolf, K. 1988, Fish viruses and fish viral diseases. Canstock Publishing Associates, Cornell University Press, Ithaca and London.). Highly virulent strains of IPNV can cause greater than 90% mortality in hatchery stocks less than four months old and survivors of infection can remain lifelong asymptomatic carriers, and serve as reservoirs of infection (McAllister, P. E., W. J. Owens, and T. M. Ruppenthal. 1987, Detection of infectious pancreatic necrosis virus in pelleted cell and particulate components from ovarian fluid of Brook trout (Salvilimus fontindis). Dis. Aquat. Org. 2:235-237). In survivors of an IPNV epizootic, the virus persists and can cause severe growth retardation in individual fish exhibiting virus persistence (McKnight and Roberts; Br. Ven. J. 132:76-86, 1976). In smolts, the virus produces considerable necrosis or inflammation of the pancreas. The virus is capable of infecting a number of different hosts and has a worldwide presence (Pilcher and Fryer. Crit. Rev. Microbial. 7:287-364, 1980).
IPNV belongs to a group of viruses called Birnaviridae which includes other bisegmented RNA viruses such as infectious bursal disease virus (chickens), tellina virus and oyster virus (bivalve mollusks) and drosophila X virus (fruit fly). These viruses all contain high molecular weight (MW) double-stranded RNA genomes. IPNV belongs to the Aquabirnavirus genus (Dobos, P. 1995, The molecular biology of infectious pancreatic necrosis virus (IPNV). Ann. Rev. Fish Dis. 5:24-54). Aquatic Birnaviruses infect marine and fresh water organisms such as fish, shrimp and other crustaceans, oysters and other mollusks.
IPNV in a brook trout hatchery was first reported in 1941(McGonigle Trans. Am. Fish Soc. 70,297, 1941). In 1960, the viral nature of the disease was confirmed (Wolf et al., Proc. Soc. Exp. Biol. Med. 104:105-110,1960). Since that time there have been isolations of the virus in a variety of fish species throughout the world, including various trout and salmon species, carp, perch, pike, eels and char, as well as mollusks and crustaceans. Acute disease has been reported primarily in a limited number of salmonid species, such as a trout and salmon.
Young fish (two-to four-month old) appear to be the most susceptible to IPNV infection, resulting in high mortality (Wolf et al. U.S. Dept. Int. Bur. Sport Fish and Wildlife, Fish Disease Leaflet 1:14, 1966; Frantsi and Savan. J. Wildlife Dis. 7:249-255, 1971). In trout, IPNV usually attacks young fry about five to six weeks after their first feeding. The affected fish are darker than usual, have slightly bulging eyes and often have swollen bellies. At the beginning of an outbreak, large numbers of slow, dark fry are seen up against water outflows, and fish are seen "shivering" near the surface. The shivering results from a characteristic symptom of the disease, a violent whirling form of swimming in which the fish rotate about their long axis. If the affected fish are examined, a characteristic white mucus is seen in the stomach. The pancreas appears to be the primary target organ for the virus, with the pancreatic fat cells or Islets of Langerhans being unaffected (McKnight and Roberts, Br. Vot. J. 132:76-86, 1976). The only organ besides the pancreas where viral lesions are consistently found is the intestine.
After an IPNV outbreak, the surviving fish generally become carriers of the virus. Trout that are carriers of the virus are a serious problem for the aqua-culture industry because the only control method currently available for eliminating the virus in carrier fish is destruction of these fish. Several factors, including age, species and water temperature, appear to influence the severity of infection and the subsequent establishment of the carrier state. Surviving carriers shed IPNV for the remainder of their lifetime (Billi and Wolf, J. Fish. Res. Bd. Can. 26:1459-1465, 1969; Yamamoto, Can. J. Micro. 21:1343-1347, 1975; Reno et al., J. Fish. Res. Bd. Can. 33:1451-1456, 1978). Therefore, IPNV is a pathogen of major economic importance to the aquaculture industry.
In view of the great deal of interest in developing a vaccine for IPNV a variety of approaches have been tried. One approach is the use of killed virus as vaccines. For example, if formalin-inactivated virus is injected intraperitoneally into four week post-hatch fry, the fish becomes immunized (Dorson, J. Virol 21:242-258, 1977). However, neither immersion of the fish into a liquid suspension of killed virus nor oral administration thereof was effective. Thus, the main problem with using killed virus is the lack of a practical method for administration of the vaccine as injection is impractical for large numbers of immature fish. Some investigators have suggested that the uptake of viral antigen by immersion might be improved if the virus was disrupted into smaller, sub-viral components, but viral disruption methods have resulted in loss of antigenicity (Hill and Way, "Serological Classification of Fish and Shellfish Birnaviruses," Abstract, First International Conference of the European Association of Pathology, Plymouth, England, 1983).
The use of attenuated viral strains has also been tried (Dorson, Abstract, International Conference on IPNV, Taloires, France, 1982). However, the earlier attenuated strains either fail to infect the fish or fail to induce protection. Strains with low virulence have been tested as vaccines for more virulent strains, but mortality from the vaccinating strain was either too high or protection was only moderate (Hill et al., "Studies of the Immunization of Trout Against IPN," in Fish Diseases, Third COPRAQ Session (W. Ahne, ed.), N.Y., pp. 29-36, 1980).
There are two distinct serogroups of IPNV, designated as serogroup A and B. Serogroup A contains 9 serotypes, whereas serogroup B contains a single serotype (Hill, B. J., and K. Way. 1995, Serological classification of infectious pancreatic necrosis (IPN) virus and other aquatic birnaviruses. Ann. Rev. Fish Dis. 5:55-77).
The IPNV genome consists of two segments of double-stranded RNA that are surrounded by a single-shelled icosahedral capsid of 60 nm diameter (Dobos, P. 1976. Size and structure of the genome of infectious pancreatic necrosis virus. Nucl. Acids Res. 3:1903-1919). The larger of the two genomic segments, segment A (3097 bp), encodes a 106-kDa polyprotein (NH2-pVP2-NS protease-VP3-COOH) which is cotranslationally cleaved by the viral protease to generate mature VP2 and VP3 capsid proteins (Dobos, P. 1977. Virus-specific protein synthesis in cells inflicted by infectious pancreatic necrosis virus. J. Virol. 21:242-258; Duncan, R., E. Nagy, P. J. Krell, and P. Dobos. 1987, Synthesis of the infectious pancreatic necrosis virus polyprotein, detection of a virus-encoded protease, and fine structure mapping of genome segment A coding regions, J. Virol. 61:3655-3664). Segment A also encodes a 15-17 kDa arginine-rich nonstructural protein (NS) from a small open reading frame (ORF) which precedes and partially overlaps the major polyprotein ORF. Although this protein is not present in the virion, it is detected in IPNV-infected cells (Magyar, G., and P. Dobos. 1994 Evidence for the detection of the infectious pancreatic necrosis virus polyprotein and the 15-17 kDa polypeptide in infected cells and of the NS protease in purified virus. Virology 204:580-589). The genomic segment B (2784 bp) encodes VP1, a 94-kDa minor internal protein, which is the virion-associated RNA-dependent RNA polymerase (Dobos, P. 1995, Protein-primed RNA synthesis in vitro by the virion associated RNA polymerase of infectious pancreatic necrosis virus. Virology 208:19-25; Duncan, R., C. L. Mason, E. Nagy, J. A. Leong, and P. Dobos, 1991, Sequence analysis of infectious pancreatic necrosis virus genome segment B and its encoded VP1 protein: A putative RNA-dependent RNA polymerase lacking the Gly-Asp-Asp motif. Virology 181:541-552). In virions, VP1 is present as a free polypeptide, as well as a genome-linked protein, VPg (Calvert, J. G., E. Nagy, M. Soler, and P. Dobos. 1991, Characterization of the VPg-dsRNA linkage of infectious pancreatic necrosis virus. J. Gen. Virol. 72:2563-2567).
Although the nucleotide sequences for genome segments A and B of various IPNV strains have been published, the precise 5'- and 3'-noncoding sequences of these strains have not been determined or confirmed (Duncan, R., and P. Dobos. 1986, The nucleotide sequence of infectious pancreas necrosis virus (IPNV) dsRNA segment A reveals one large ORF encoding a precursor polyprotein. Nucl. Acids Res. 14:5934; Duncan, R., C. L. Mason, E. Nagy, J. A. Leong, and P. Dobos, 1991, Sequence analysis of infectious pancreatic necrosis virus genome segment B and its encoded VP1 protein: A putative RNA-dependent RNA polymerase lacking the Gly-Asp-Asp motif. Virology 181:541-552; Havarstein, L. S., K. H. Kalland, K. E. Christie, and C. Endresen, 1990, Sequence of large double-stranded RNA segment of the N1 strain of infectious pancreatic necrosis virus: a comparison with other Birnaviridae. J. Gen. Virol. 71:299-3908). Unlike IBDV, there is extensive homology between the noncoding sequences of IPNV segments A and B. For example, 32 of 50 nucleotides at the 5'-noncoding region and 29 of 50 nucleotides at the 3'-noncoding region between the two segments are conserved. These termini should contain sequences that are important in packaging and replication of IPNV genome, as demonstrated for other double-stranded RNA viruses such as mammalian reoviruses and rotaviruses (Gorziglia, M. L. and P. L. Collins. 1992, Intracellular amplification and expression of a synthetic analog of rotavirus genomic RNA bearing a foreign marker gene: Mapping cis-acting nucleotides in the noncoding region. Proc. Nati. Acad. Sci. USA 89:5784-5788; Patton, J. T., M. Wentz, J. Xiaobo, and R. F. Ramig. 1996, cis-Acting signals that promote genome replication in rotavirus mRNA. J. Virol. 70:3961-3971; Wentz, M. J., J. T. Patton, and R. F. Ramig. 1996. The 3-terminal consensus sequence of rotavirus mRNA is the minimal promoter of negative-strand RNA synthesis. J. Virol. 70:7833-7841; Zou,S., and E. G. Brown. 1992. Identification of sequence elements containing signals for replication and encapsulation of the reovirus M1 genome segment. Virology 186:377-388).
In recent years, a number of animal RNA viruses have been recovered from cloned cDNA, such as polio virus (a plus-stranded RNA virus), influenza virus (a segmented negative-stranded RNA virus), and rabies virus (a nonsegmented negative-stranded RNA virus) (Enami, M., W. Luytjes, M. Krystal, and P. Palese. 1990. Introduction of site-specific mutations into the genome of influenza virus. Proc. Natl, Acad. Sci. USA 87:3802-3807; Racaniello, V. R., and D. Baltimore. 1981. Cloned poliovirus complementary DNA is infectious in mammalian cells. Science 214:916-919; Schnell M. J, T. Mebatsion, and K. K. Conzelmann. 1994, Infectious rabies viruses from cloned cDNA. EMBO J. 13:4195-4205). However, to date, there is no report of a recovered infectious dsRNA virus of aquatic species.
One of the present inventors recovered a virus of segmented dsRNA genome from synthetic RNAs only. The reverse genetics system for birnavirus was developed by one of the present inventors who demonstrated that synthetic transcripts of infectious bursal disease virus (IBDV) genome are infectious (Mundt, E., and V. N. Vakharia. 1996, Synthetic transcripts of double-stranded birnavirus genome are infectious. Proc. Natl. Acad. Sci. USA 93:11131-11136).
In order to develop a reverse genetics system for IPNV, full-length cDNA clones of segments A and B of the West Buxton and SP strains were constructed. Complete nucleotide sequences of these cDNA clones were determined, including the 5'- and 3'-noncoding regions. Furthermore, one of the cDNA clones was modified by site-directed mutagenesis to create a tagged sequence in segment B. Synthetic plus-sense RNA transcripts of segments A and B were produced by in vitro transcription reactions on linearized plasmids with T7 RNA polymerase, and used to transfect chinook salmon embryo (CHSE) cells. In this application, the recovery of IPNV from CHSE cells transfected with combined RNA transcripts of segments A and B is described.
In order to study the function of NS protein in viral pathogenesis, the present inventors constructed a cDNA clone of IPNV segment A, in which the initiation codon of the NS gene was mutated to prevent the expression of the NS protein. Using the reverse genetics system, a wild-type IPNV was generated, as well as a mutant IPNV that lacked the expression of the NS protein. The properties of the recovered wild-type IPNV and mutant IPNV in cell culture were compared and their pathological function in the host evaluated.